The long range goal of the proposed research is to develop strategies for the chemical synthesis of ion channel proteins and to use chemical synthesis to investigate the mechanisms of ion channel function. Understanding the relationship between the atomic structure of a protein and the biological function requires the ability to perturb the protein structure in a precise manner. Chemical synthesis facilitates the incorporation of a wide variety of side chain and peptide backbone modifications that enables precise modifications of the structural and electronic properties of the protein. Similar modifications are not possible using conventional mutagenesis making chemical synthesis an important asset in investigations of protein structure and function. The size of the protein has been a major factor limiting the use of chemical synthesis to investigate proteins. In the field of membrane proteins, chemical synthesis has so far been accomplished only for relatively small (< 150 amino acids) proteins. Proteins of interest such as voltage gated ion channels are much bigger and are presently not amenable to chemical synthesis. To overcome this limitation, we propose developing methods that can be used for the chemical synthesis of large (> 150 amino acid) membrane proteins thereby enabling us to use chemical synthesis to investigate these proteins. We will pursue three major specific aims. Aim 1) To develop a chemical synthesis of the voltage gated K+ channel KvAP, and the non-selective channel, NaK. The chemical synthesis protocols that we develop in this aim will be useful not only in investigating these specific proteins but will also find applicability in the chemical synthesis of other important classes of membrane proteins. Aim 2) We will investigate the slow inactivation process in the KvAP channel. Understanding the process of slow inactivation is important because the rate of entry (and exit) of channels into the inactivated state can significantly alter the number of channels available and therefore the electrical properties of the cell. Aim 3) We will investigate the binding of divalent ions to the outer vestibule of the NaK channel using chemical synthesis. The NaK channel shows sequence and functional similarity to cyclic nucleotide gated ion channels. Therefore, the mechanisms that we uncover in our investigations of the NaK channel will be relevant in understanding the physiologically important interactions of divalent ions with CNG channels. PUBLIC HEALTH RELEVANCE: Ion channel function underlies many important biological processes such as the excitation of nerve and muscle cells, the secretion of hormones, and sensory transduction. The research proposed is significant because it will provide a deeper understanding of the physiologically important processes of slow inactivation and block by divalent ions. Further, the research is important as it will establish chemical synthesis as an important tool for understanding ion channel structure and function.